The overall goal of this research is to understand the interactions between spin probes and paramagnetic metal ions when the dual probe technique is used to investigate the structure and function of biological systems. Nitroxyl radical EPR and relaxation times will be studied as a function of the identity and concentration of the metal species, identity of buffers or other salts present and the size of the nitroxyl and metal species. These experiments will be compared with predictions based on the theory of dipolar and exchange interactions. The functional dependence for particular variables in the theoretical expressions which fit the experimental results will be used as a guide to optimization of that parameter. Simultaneous with application of previously published expressions for relaxation times and line broadening, a less approximate treatment will be derived using a density matrix approach. The results of these small molecule studies will be used to guide the selection of optimally effective dual probe studies of membranes and metalloproteins. The first metalloprotein chosen for study is plastocyanin, which is sufficiently well characterized that it can serve as a calibrant of the ideas on metal-nitroxyl interaction. Lipid bilayer model membranes will be used to study the effect of paramagnetic metals on spin probes used to estimate interval viscosity in cells and in membranes. These studies will build toward exploration of the very complicated membrane of thylakoids.